PROCESS FOR PREPARATION OF SECRETORY IgA AND SECRETORY IgM

ABSTRACT

A process for synthesizing and separating secretory IgA from a mixture of IgA monomer and IgA dimer is provided. The process includes covalently binding affinity tagged or epitope tagged recombinant secretory component to the IgA dimer in the mixture and binding the affinity tagged or an epitope tagged secretory IgA to immobilized moieties on the solid phase support resin to which the affinity tag or epitope tag binds and then eluting the affinity tagged or an epitope tagged secretory IgA with release buffer. A process for synthesizing and separating secretory IgM from a mixture of IgM and other plasma proteins is also provided. The process includes covalently binding affinity tagged or an epitope tagged recombinant secretory component to the IgM in the mixture and binding the affinity tagged or an epitope tagged secretory IgM to immobilized moieties on the solid phase support resin and then eluting the peptide tagged secretory IgM with a release buffer.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 13/935,417; filed Jul. 3, 2013; and a continuation-in-part of PCT application Serial No. PCT/EP2013/054697 filed Mar. 8, 2013 that in turn claims priority benefit of EP 12158931.1 filed Mar. 9, 2012 and EP 12168343.7 filed May 16, 2012; the contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates in general to a process for the preparation of secretory IgA and secretory IgM from plasma proteins containing IgA and/or IgM, and in particular to a process that is scalable to allow the production of commercial quantities of medicaments containing the same.

BACKGROUND OF THE INVENTION

Clostridium difficile (C. difficile) is a gram-positive anaerobic bacillus. Disease is associated with C. difficile infection.

Antibiotic associated pseudomembranous colitis results from the use of broad-spectrum antibiotic agents such as clindamycin. These antibiotics cause diarrhea in about 10% of treated patients and pseudomembranous colitis in about 1%. C. difficile causes antibiotic associated diarrhea and almost all cases of pseudomembranous colitis.

Pseudomembranous colitis results from the production of C. difficile toxin A (MW, 308,000) and toxin B (MW, 270,000) in the colon (Barroso et al., Nucleic Acids Res., 18:4004; Dove et al., Infect. Immun., 58:480-488; Lyerly et al., Clin. Microbiol. Rev., 1:1-18). Toxin A probably causes most of the gastrointestinal symptoms because of its enterotoxic activity (Lyerly et al., Infect. Immun., 35:1147-1150; Lyerly et al., Infect. Immun., 47:349-352). The toxins may act synergistically and the initial pathology caused by toxin A allows toxin B to manifest its toxicity (Lyerly et al., Infect. Immun., 47:349-352).

Most patients with C. difficile associated disease are treated effectively with vancomycin or metronidazole. Other treatment modalities include tolevemer, a toxin binding polymer (T. J. Louie et al., Clin. Infect. Dis. 2006; 43:411), and an antiparasitic medication, nitazoxanide (Med. Letter Drugs Ther. 2006; 48:89). Current treatments for C. difficile associated disease use antibiotics such as metronidazole and vancomycin. These drugs result in further disruption of the intestinal flora and are associated with a 20-25% incidence of disease relapse. Therefore, there is still a need for additional treatments of Clostridium difficile associated disease in humans.

Immunological treatment is valuable against C. difficile. Vaccination against toxins A and B stimulates active immunity against C. difficile disease in animals (Libby et al., Infect. Immun., 36:822-829).

Passive immunization is another immunological treatment against C. difficile. Serum antibodies against C. difficile have been shown to protect hamsters against C. difficile disease after oral administration. Passive immunization with bovine antibodies has been proposed as a treatment for other infectious diseases of the gastrointestinal tract, such as diseases caused by rotavirus, enteropathogenic and enterotoxigenic Escherichia coli, Vibrio cholerae, and Cryptosporidium parvum. Preliminary studies indicate that such passive immunization provides protection (Boesman-Finkelstein et al., Infect. Immun., 57:1227-1234; Brussow et al., J. Clin. Microbiol., 25:982-986; Fayer et al., Infect. Immun., 58:2962-2965; Hilpert et al., J. Infect. Dis., 156:158-166; Mietens et al., Eur. J. Pediatr., 132:239-252; Tacket et al., N. Engl. J. Med., 318:1240-1243; Yoshiyama et al., Immunology, 61:543-547).

It has been reported that bovine immunoglobulin G (IgG) concentrate from the colostrum of cows vaccinated with C. difficile toxoid protects hamsters against antibiotic associated cecitis. The hamsters were protected when treated before the onset of diarrhea but not after diarrhea began (Lyerly et al., Infection and Immunity, Vol. 59, No. 6, pages 2215-2218 (1991)). IgG directed against toxins A and B of C. difficile are present in the general population (Bacon and Fekety, Diagn. Microbiol. Infect. Dis., 1994; 18:205-209). Human intravenous immunoglobulin derived from plasma donors has facilitated treatment in some patients, especially patients who lack circulating antibodies to the C. difficile toxins (Leung D. Y. et al., J. Pediatr. 1991 April; 118(4 (Pt 1)):633-7; Salcedo J. et al., Gut 1997; 41:366-370; Wilcox M. H., J. Antimicrob. Chemoth. 2004; 53:882-884; McPherson S. et al., Dis. Colon Rectum 2006; 49:640-645; Cone L. A. et al., Infect. Dis. Clin. Pract. 2006; 14:217-220).

In vitro experiments have demonstrated that polymeric IgA is superior to monomeric IgA and IgG in preventing C. difficile toxin damage to intestinal epithelial cell monolayers (Stubbe H. et al., J. Immunol. 2000; 164:1952-1960). Selective neutralization of C. difficile toxin by serum IgA has also been demonstrated (Johnson S. et al., Infect. Immun. 1995; 63:3166-3173).

Administration of an immunoglobulin product containing specific antibodies to C. difficile results in the elimination of C. difficile toxins and also killing of the bacteria within the colon as detailed in U.S. Pat. No. 5,773,000. Such passive immunization therefore provides an effective approach for the treatment of C. difficile associated diseases such as colitis, pseudomembranous colitis and antibiotic associated diarrhea. This is especially important for patients experiencing multiple relapses.

Oral human immunoglobulin treatment has shown efficacy in treating acute C. difficile infections. Monomeric IgA admixed with IgG (2:1) was derived from plasma (IgAbulin, Immuno, Vienna) (100 mg/mL); four mL administered orally 3 times daily for 3 weeks to a three and one-half year old child with antibiotic-associated diarrhea and C. difficile toxin A in his stools proved effective in the context of concurrent vancomycin administration. The child improved on this treatment (Tjellstrom B. et al., Lancet 1993; 341:701702). This report demonstrates the efficacy of passive immunization with IgA derived from the general population.

The isolation of secretory forms of immunoglobulins is more difficult that securing forms of a respective immunoglobulin that circulate in the blood and lack secretory component.

Thus, there exists a need for IgA and IgM therapeutics that are resistant to gastrointestinal tract degradation. There also exists a need to provide such a therapeutic in a dosing form well suited for treating an infected subject. Human plasma derived IgA and IgM has been successfully combined with recombinant secretory component to produce secretory IgA and secretory IgM with biological activity (Longet et al 2013).

SUMMARY OF THE INVENTION

A process for the preparation of secretory IgA and/or secretory IgM and their separation from unwanted other substances including proteins is provided. The process involves the application of an IgA source, which may contain a mixture of monomers and dimers, or a plasma protein solution containing IgM, to secretory component that is modified to contain an affinity tag or an epitope tag to form secretory IgA and/or secretory IgM containing said affinity tag or epitope tag that is useful for capture by a solid phase support resin. The protein solution which now contains the affinity tagged or epitope-tagged secretory IgA or affinity tagged or epitope-tagged secretory IgM is then applied to a solid phase support resin. The adherence of the affinity tagged or epitope tagged secretory IgA or secretory IgM to the resin and the unwanted components flow through and are thus removed. The desired product, secretory IgA or secretory IgM is then eluted from the solid phase support resin using a release agent.

For histidine and “FLAG” peptides, examples of the moieties on the solid phase support resin to which the peptide binds are immobilized divalent cations or anti polyhistidine antibodies and immobilized anti-FLAG peptide antibodies. A new composition of histidine tagged secretory IgA or secretory IgM is also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. ELISA Δ colostral secretory IgA (sIgA) and □ synthesized sIgA. Plate was coated with mouse anti-secretory component antibody.

FIG. 2. Tris acetate PAGE showing sIgA, prepared with histidine tagged secretory component eluted from nickel resin (Cube Biotech, Monheim, Germany). Lane 1: MW ladder; Lane 2: colostral secretory IgA; Lane 3 blank; Lane 4 nickel resin flow-through; Lane 5: nickel resin imidazole eluate with semisynthetic sIgA.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention has utility for the preparation of secretory IgA or secretory IgM. In some inventive embodiments, the IgA is derived from a mixture of monomeric and dimeric plasma IgA. or in other inventive embodiments, the preparation of secretory IgM is derived from a mixture of IgM with other plasma proteins. The present invention is superior to monomeric IgA or pentameric IgM administered orally because the presence of secretory component protects the IgA or IgM from digestion in the gastrointestinal tract. Without intending to be bound to particular theory, it is believed that the increased efficacy of the present invention is achieved for secretory IgA or secretory IgM owing to the propensity of monomeric IgA and pentameric IgM to degrade in the gastrointestinal tract. The resultant dosing requirements increase treatment costs. While the present invention is further detailed principally with respect to IgA, it is appreciated that the process and medicaments that result are equally applicable to IgM and the resulting secretory IgM, regardless of whether the tag is retained or removed.

An affinity tag or an epitope tag that are efficaceous for the present invention is one of: peptide tags:AviTag, a peptide allowing biotinylation by the enzyme BirA so the protein can be isolated by streptavidin; GLNDIFEAQKIEWHE (SEQ ID No. 1); calmodulin-tag, a peptide bound by the protein calmodulin (KRRWKKNFIAVSAANRFKKISSSGAL (SEQ ID No. 2); FLAG-tag, a peptide recognized by an antibody DYKDDDDK (SEQ ID No. 3); Hemaglutinin-tag, a peptide recognized by an antibody YPYDVPDYA (SEQ ID No. 4); His-tag, 5-10 histidines bound by a nickel or cobalt or other divalent cation chelate HHHHHH (SEQ ID No. 6); Myc-tag, a short peptide recognized by an antibody EQKLISEEDL (SEQ ID No. 7); S-tag KETAAAKFERQHMDS (SEQ ID No. 8); SBP-tag, a peptide which binds to streptavidin MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP (SEQ ID No. 9); Softag 1, for mammalian expression SLAELLNAGLGGS (SEQ ID No. 10); Softag 3, for prokaryotic expression TQDPSRVG (SEQ ID No. 11); V5 tag, a peptide recognized by an antibody GKPIPNPLLGLDST (SEQ ID No. 12); Xpress tag DLYDDDDK (SEQ ID No. 13); Biotin Carboxyl Carrier Protein, a protein domain recognized by streptavidin; Glutathione-S-transferase-tag, a protein which binds to immobilized glutathione; Green fluorescent protein-tag, a protein which is spontaneously fluorescent and can be bound by nanobodies; Maltose binding protein-tag, a protein which binds to amylose agarose; Nus-tag; Strep-tag, a peptide which binds to streptavidin, or the modified streptavidin called streptactin Strep-tag II: WSHPQFEK (SEQ ID No. 14); Thioredoxin-tag; TC tag; or Ty tag.

Plasma IgA contains a mixture of monomer and dimer (Delacroix et al. 1981; Delacroix et al. 1983; Longet et al. 2013). In some embodiments of the present invention, plasma dimeric IgA in the naturally occurring monomer-dimer mixture is covalently bound to recombinant peptide tagged secretory component in vitro. In other inventive embodiments native secretory component is covalently bonded to one or more amino acid residues through conventional synthetic techniques (Hermanson G T 1996). As an example using a histidine tag, it is appreciated that a single histidine residue or a poly histidine having typically between 2 and 20 histidine residues is added to secretory component, regardless of whether produced by recombinant, synthetic addition, or other technique. The secretory IgA is now histidine tagged by virtue of the divalent bonding of the histidine tagged secretory component to the IgA dimer, The novel method of obtaining purified secretory IgA that is thus formed is to remove the secretory IgA that is now tagged by affinity binding of one of the aforementioned tags to a binding moiety immobilized on a resin a or simply by a histidine residue(s) tagged to an immobilized nickel⁺² resin. Alternatively, other immobilized divalent metal ions such as cobalt, zinc, copper or iron can be used. Alternatively, a FLAG peptide is used in certain inventive embodiments and antibody to the FLAG peptide is immobilized on the solid support resin. FLAG tags have been detailed elsewhere as for example U.S. Pat. No. 4,703,004. The resultant secretory IgA has utility, for example, as a treatment of C. difficile associated diseases such as Clostridium difficile colitis, pseudomembranous colitis and antibiotic associated diarrhea and in particular to secretory immunoglobulin A (IgA) compositions administered in the form of pharmaceutical compositions. The above process is equally applicable to IgM to form purified secretory IgM.

A more detailed description of an exemplary isolation of an IgA component as a byproduct from pooled human plasma or hyperimmune pooled human plasma is as follows. Ethanol fractionation of pooled human plasma is a well-known process to prepare immunoglobulin G. Pooled human plasma is first obtained from licensed plasmapheresis centers in the United States and tested for various pathogens including the HIV virus. The first manufacturing step of most commercial immunoglobulin G preparations involves a modified cold ethanol fractionation according to Cohn to produce Cohn fraction II. In the fractionation process, many infectious viruses are eliminated from the pooled human plasma. Following fractionation, the Cohn fraction II is subjected to adsorption onto an ion exchange medium. This step may selectively reduce the IgA concentration to less than 0.1%. Such a step is important for producing immunoglobulin G for intravenous infusion into humans. This is because some individuals undergo an anaphylactic-like reaction if treated with intravenous IgG that contains IgA as an impurity.

The modified cold ethanol fractionation process according to Cohn is a series of fractionations using various levels of ethanol, pH, and temperature to produce a fraction II which is further treated to produce immunoglobulins as described above. In the fractionation process, pooled human plasma is first treated to produce a cryoprecipitate and cryo-supernatant. Alternatively, it is appreciated that the source plasma may be autologous plasma or hyperimmune human plasma, either pooled, or from a single individual who has been immunized against a specific disease.

In another embodiment, the IgA component is be prepared by hybridoma techniques to provide antigen-specific IgA. Hybridoma techniques are described originally in Kohler and Milstein, Nature 1975; 256:495-497 with more recent advances summarized in Berzofsky et al., Fundamental Immunology, Third Edition, 1993, pp 455-62.

Regardless of the source, the cryo-supernatant is subjected to a first ethanol fractionation to yield a supernatant I. Supernatant I is subjected to a second ethanol fractionation to yield fraction II+III. Fraction II+III is subjected to a third ethanol fractionation procedure to yield a supernatant III and Fraction III precipitate.

The fraction III precipitate enriched in IgA is generally discarded as an unwanted byproduct. According to the present invention, this unwanted IgA following ion exchange adsorption purification is further treated by incubation with immobilized hydrolases to inactivate viruses and vasoactive substances. Such treatment has been proven to eliminate many viruses tested including HIV, Sindbis, and vaccinia. Other antiviral treatments, as known to those skilled in the art, are used in combination and consist of solvent detergent processes, nanofiltration and/or heat inactivation. Usually three antiviral steps are implemented. Following incubation to remove viruses, the concentration of the active material is adjusted with sterile saline or buffered solutions to ensure a constant amount of active material per milliliter of reconstituted product. Finally, the solution with a constant amount of reconstituted product is sterilized by filtration before use.

The ethanol fractionation process according to Cohn is well known in the art and is described in Cohn et al., J. Am. Chem. Soc. 1946; 68:459-475, Oncley et al., J. Am. Chem. Soc. 1949; 71:541-550, and in most detail in pages 576-602, Kirk-Othmer Encyclopedia of Chemical Technology, Vol. 3, second edition (1963). Alternatively, ion exchange chromatography may be used to obtain the dimeric and polymeric IgA byproduct during the manufacture of intravenous immunoglobulin. From 4% to 22% of plasma IgA is dimeric and polymeric IgA (Delacroix et al. 1981; Delacroix et al. 1983). The resulting dimeric IgA-J chains are purified to form a medicament.

The dimeric and polymeric IgA present in the plasma IgA monomer-polymer mixture is further coupled to secretory component that is recombinantly produced to include a histidine tag or another of the aforementioned tags; or subsequently covalently bonded to a peptide tag such as histidine or poly-histine oligopeptide. In certain inventive embodiments, the coupling of IgA to secretory component is accomplished by forming disulfide bonds under mildly oxidizing conditions. (Jones R. M. L., Schweikart F., Frutiger S., Jaton J-C., Hughes G. J. Thiol-disulfide redox buffers maintain a structure of immunoglobulin A that is essential for optimal in vitro binding to secretory component. Biochimica et Biophysica Acta 1998; 1429:265-274.) Dimeric and polymeric IgA containing both J chain and secretory component is purified from the mixture by immobilized metal ion affinity chromatography, as performed by those of skill in the art of protein purification.

It has been previously found that it is possible to separate recombinant proteins from cell supernates by producing such proteins with histidine tails or other of the aforementioned tags and then passing the cell supernates through nickel bound solid support resins. The histidine or other tag adheres to the nickel or other suitable tag specific binding moiety and is retained while the unwanted proteins are washed through. The tagged secretory immunoglobulin protein is then recoverd by eluting it with an imidazole buffer in the case of an amide-metal bond between target protein and resin (Block H et al 2009).

The mixture of histidine tagged secretory IgA and residual monomeric IgA is buffer exchanged into a binding buffer containing low concentrations of imidazole (≦40 mM). Another release agent operative to exchange histidine tagged secretory IgA or secretory IgM illustratively includes 1) ethylene diamine tetraacetic acid (EDTA) at 10 mM and 2) an elution buffer of pH 5.5 or lower. Typical binding buffer imidazole concentrations range from 0.1 to 100 millimolar (mM). It is appreciated that the initial binding buffer pH is somewhat variable and readily discerned for a given chemical structure of buffer and concentration through routine experimentation. The chromatography medium operative herein is selected to be stable in the presence of the binding buffer and able to separate histidine tagged secretory IgA. Suitable media illustratively include Exemplary of these media are nickel, cobalt and zinc immobilized on sepharose. In a preferred embodiment, the affinity medium is washed in a wash buffer containing from 0 to 100 mM imidazole to remove unbound monomeric IgA. The bound histidine tagged secretory IgA is recovered using an elution buffer of a higher imidazole concentration (e.g. 100 to 1000 mM). With succesive elutions separation of monomeric from histidine tagged secretory component bound dimeric IgA is exacted. It is appreciated thate the inventive process is amenable to scaling to produce quantities sufficient to treat numerous subjects. It is appreciated that similar selective binding pairs is achieved between other inventive tagged secretory component containing immunoglobulin proteins and resins are convetional to the art for each of the aforementioned tags.

By way of a specific example, the binding and wash buffers are 50 mM NaH₂PO₄ 300 mM NaCl and 20 mM imidazole, is adjusted to pH 8. The mixture of IgA monomer and secretory IgA is dissolved in that buffer. The elution buffer is identical to the binding buffer with the exception that the imidazole is at a higher concentration, e.g 100 to 1000 mM.

The remaining histidine tagged secretory IgA is then eluted from the divalent immobilized metal resin with the elution buffer according to conventional techniques and conditions that include an exemplary basic pH of for example 8 to 10, see FIGS. 1 and 2.

Purified secretory IgA containing histidine tagged secretory component is stabilized in some embodiments for example by the addition of human serum albumin to a final concentration of 5% total weight albumen.

In another embodiment, the tag is removed from the recovered secretroy IgA and native secretory IgA is available for usage as a medicament. For a histidine tagged secretoty IgA a procedure for tag removal is known to the art (Kopera E et al 2012).

In summary, the inventive process is the addition of tagged secretory component in either recombinatant or post expression tagging to a mixture of plasma derived IgA monomers and dimers, in which the tagged secretory component combines with the IgA dimer forming secretory IgA and allows recovery of the newly formed secretory IgA by adhesion to immobilized divalent metal ions or other solid phase moeity, and subsequent elution therefrom.

Plasma IgM can be recovered from the byproducts of the production of intravenous immunoglobulin. An example of such a byproduct is Cohn fraction III preciptate. The IgM is most easily solublized from Cohn fraction III preciptate by 20 mM socium acetate. Other plasma proteins are similarly solublized along with the IgM. The plasma IgM in this protein mixture is covalently bound to recombinant histidine tagged secretory component in vitro forming secretory IgM within the protein mixture. The secretory IgM is now tagged by virtue of the divalent bonding of the tagged secretory component to the IgM. The novel method of obtaining purified secretory IgM that is thus formed is to remove the secretory IgM that is now tagged by affinity binding of the tag to a immobilized nickel⁺² or other divalent metal ion or other suitable binding moiety conventional to the art that is part of a resin column. The resultant semisynthetic secretory IgM has utility, for example, as a treatment of Clostridium difficile associated diseases such as Clostridium difficile colitis, pseudomembranous colitis and antibiotic associated diarrhea and in particular to secretory immunoglobulin M compositions administered in the form of pharmaceutical compositions.

In another embodiment, the tag is removed from the recovered secretory IgM and native secretory IgM is available for usage as a medicament. For a histidine tagged secretoty IgM a procedure for tag removal is known to the art (Kopera E et al 2012).

Thus, an inventive process provides the addition of peptide tagged secretory component to a mixture of plasma derived IgM and other plasma proteins, in which the peptide tagged secretory component combines with the IgM forming secretory IgM and allows recovery of the newly formed secretory IgM by adhesion to moieties on the solid phase support resin to which the peptide binds, and subsequent elution therefrom using an elution buffer.

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Patent applications and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. These applications and publications are incorporated herein by reference to the same extent as if each individual application or publication was specifically and individually incorporated herein by reference.

The foregoing description is illustrative of particular embodiments of the invention, but is not meant to be a limitation upon the practice thereof. The following claims, including all equivalents thereof, are intended to define the scope of the invention. 

1. A process for synthesizing and separating an immunoglobulin of secretory IgA from an a monomer and dimer mixture of either IgA mixed with other proteins comprising: adding secretory component tagged with an affinity tag to the protein mixture containing IgA dimers, facilitating the covalent binding of the affinity tagged secretory component to the IgA dimer; recovering the secretory IgA by adhesion of the affinity tagged secretory IgA to immobilized moieties on the solid phase support resin to which the affinity tag or epitope tag binds; and eluting the bound secretory IgA with a release buffer.
 2. The process of claim 1 wherein the secretory component is human.
 3. The process of claim 1 wherein the monomer and dimer mixture is obtained from human plasma.
 4. The process of claim 1 wherein the peptide tag is removed from the secretory IgA.
 5. The process of claim 1 wherein the peptide tag is histidine or polyhistidine.
 6. The process of claim 1 wherein the moiety on the solid phase support resin to which the peptide binds is a divalent cation.
 7. The process of claim 1 further comprising washing the unbound IgA monomer through the resin prior to the eluting the secretory IgA.
 8. The process of claim 1 wherein the secretory component is recombinant.
 9. The process of claim 8 wherein the secretory component is expressed with said histidine or polyhistidine.
 10. The process of claim 1 further comprising coupling said histidine or polyhistidine to the secretory component in vitro.
 11. A composition comprising: a dimeric IgA; secretory component bonded to said dimeric IgA; and a histidine or polyhistidine covalently bonded to said secretory component.
 12. A process for synthesizing and separating a secretory IgM immunoglobulin from pentameric IgM mixed with other proteins comprising: adding secretory component tagged with an epitope tag to the protein mixture containing IgM pentamers, facilitating the covalent binding of the epitope tagged secretory component to the IgM pentamer; recovering the secretory IgM by adhesion of the epitope tagged secretory IgM to immobilized moieties on the solid phase support resin to which the affinity tag or epitope tag binds; and eluting the bound secretory IgM with a release buffer.
 13. The process of claim 12 wherein the secretory component is human.
 14. The process of claim 12 wherein the mixture is obtained from human plasma.
 15. The process of claim 12 wherein the epitope tag is removed from secretory IgM.
 16. The process of claim 12 wherein the moiety on the solid phase support resin to which the epitope tag binds is a divalent cation.
 17. The process of claim 12 further comprising washing the unbound IgM monomer through the resin prior to the eluting the secretory IgM.
 18. The process of claim 12 wherein the secretory component is recombinant.
 19. The process of claim 12 further comprising coupling said histidine or polyhistidine to the secretory component in vitro.
 20. A composition comprising: a pentameric IgM; secretory component bonded to said pentameric IgM; and a histidine or polyhistidine covalently bonded to said secretory component. 